WATER BUFFALO MICROBIOTA AND IMMUNITY DURING INFECTIOUS DISEASES

The thesis aims to investigate the immune response of water buffaloes (Bubalus bubalis) during infectious diseases (e.g. mastitis, brucellosis and tuberculosis) and productions diseases, such as peripartum related metabolic diseases or stress. Given the relationship between the microbial environment and the immune system, the microbiota content of milk has been identified as well. The immune system of water buffalo has been poorly addressed so far, not to mention the microbiota, which was unknown. Dairy water buffaloes are sensitive to the same diseases as dairy ruminants, such as for example mastitis, tuberculosis, brucellosis, but the impact of these diseases on water buffaloes’ immune system are unknown. Water buffalo presents anatomical (e.g. at mammary gland and skin level) and physiological (peripartum and infectious diseases) differences as compared to cow and other dairy ruminants. Therefore, it is evident that both the immune system and microbiota could present several differences. In my PhD thesis, I tried to address some of these issues. Firstly, the milk microbiota of water buffaloes was investigated in relation to mastitis disease. Secondly, the evaluation of the immune response, in terms of gene expression ad miRNAs, was carried out in animals affected by brucellosis and tuberculosis. Finally, the characterization of the transition period was performed measuring acute phase proteins to assess the inflammation status during the transition period. The water buffalo milk microbiota was described herein for the first time. The variation in composition and structure was demonstrated depending on the pathological status of the animals. A change in milk microbial community was observed in clinical and subclinical mastitis-affected quarter milk samples as compared with a health condition, as well as a reduction of microbiota diversity in mastitis-affected quarters. The treatment of subclinical mastitis quarters using inactivated cultures of Lactobacillus rhamnosus was performed in order to unravel the effect of alternative strategies to the therapeutic use of antibiotics. A chemotactic response at quarter level was observed, but no change neither in milk microbiota nor in microbiological culture was found after treatment, suggesting that the use of pro- and pre-biotic for mastitis treatment needs to be further investigated. Given that, so far and in cow, each udder quarter has been regarded as an entity not related to the other three, the hypothesis of the presence of interdependence within milk udder quarters was demonstrated, studying the intra- and inter-individual variation in milk microbiota. Individual signatures may explain the higher similarity between milk microbiota of quarters within the same udder, suggesting that the independence of milk quarters has been challenged and needs to be taken into account when performing microbiota studies. Finally, in order to improve the taxonomic resolution of the experimental design, milk microbiota was characterized using a longer amplicon sequencing by using the full-length 16S marker ( ̴ 1500pb) taking advantage of Nanopore technology, that confirmed the higher resolution power as compared with short-read sequencing ( ̴ 250pb) and could provide a supportive role for microbiological culture technique. The investigation of a possible biomarker for brucellosis in vaginal fluid and serum revealed that levels of 10 miRNAs were modulated in vaginal secretion of seropositive compared to seronegative animals. Furthermore, the diagnostic accuracy of the combination of six miRNAs was good with a sensitivity of 95.45 and a specificity of 85, suggesting that a multimiRNAs panel would allow an early and fast procedure for brucellosis diagnosis. A modification in the immune response during tuberculosis disease was observed, aiming to address the main issue of false-positive during routine TB diagnosis. An unbalanced of cytokines expression toward Th1 reaction was found in M. bovis positive animals after intradermal tuberculin test as compared to those M. avium positives. The positive correlation of the expression of IFNg with two transcriptional factors related to Th1 polarization (TBET and STAT4) demonstrates the pro-inflammatory activity present in animals with tuberculosis. Acute phase proteins (APPs) were also found to be modulated during peripartum, An increase of Serum Amyloid A (SAA) and haptoglobin (Hp) were observed in the first week after parturition, while the increase of the a1-acid glycoprotein (AGP) concentrations from prepartum to early lactation was later than for Hp and SAA and was also not limited to a peak value but was continued until 6 weeks post-partum.